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Separating and purifying method for thrombase

A technology for separation and purification of thrombin, applied in the field of bioengineering, can solve the problems of high cost, decreased thrombin yield and high fibrinogen content

Inactive Publication Date: 2005-03-02
SHAANXI UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process is simpler, but uses affinity chromatography, which is more expensive
In addition, the fibrinogen content in the crude product of prothrombin obtained by the above two methods is too high, and the loss of thrombin after activation leads to a decrease in the yield of thrombin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0006] Embodiment 1: at first get animal blood as raw material, add the sodium citrate solution that mass percent concentration is 3.8% as anticoagulant in this blood by the volume ratio of 9: 1; Centrifuge for 10 minutes to separate the plasma; freeze the plasma at -30°C for 1 day, thaw at 2°C, and centrifuge at 2600 rpm at 4°C for 7 minutes to separate the supernatant; add to each liter of supernatant 80ml of BaCl with a concentration of 1mol / l 2 solution, centrifuge at 4°C at 4000 rpm for 10 minutes to collect the precipitate; add 0.2 mol / l EDTA solution with a pH value of 7.4 to the precipitate for desorption, and centrifuge at 4°C at 3800 rpm Centrifuge for 5 minutes to discard the precipitate and obtain the crude prothrombin solution; add CaCl to the crude prothrombin solution at room temperature 2 The solution makes the concentration of the crude prothrombin solution reach 0.1 mol / l, and then the pure thrombin product is obtained through DEAE-cellulose anion exchange c...

Embodiment 2

[0007] Embodiment 2: first get animal blood as raw material, in this blood, add the sodium citrate solution that mass percentage concentration is 3.8% by the volume ratio of 9:1 as anticoagulant; Centrifuge for 7 minutes to separate the plasma; freeze the plasma at -25°C for 3 days, thaw at 4°C, and centrifuge at 2800 rpm for 8 minutes at 4°C to separate the supernatant; add to each liter of supernatant 90ml of BaCl with a concentration of 1mol / l 2 Solution, centrifuge at 3,500 rpm at 4°C for 6 minutes to collect the precipitate; add 0.2 mol / l EDTA solution with a pH value of 7.4 to the precipitate for desorption, and centrifuge at 4,000 rpm at 4°C Centrifuge for 9 minutes to discard the precipitate and obtain the crude prothrombin solution; add CaCl to the crude prothrombin solution at room temperature 2 The solution makes the concentration of the crude prothrombin solution reach 0.1 mol / l, and then the pure thrombin product is obtained through DEAE-cellulose anion exchange ...

Embodiment 3

[0008] Embodiment 3: first get animal blood as raw material, in this blood by the volume ratio of 9: 1, add the sodium citrate solution that mass percentage concentration is 3.8% as anticoagulant; Centrifuge for 5 minutes to separate the plasma; freeze the plasma at -29°C for 3 days, thaw at 3°C, centrifuge at 4°C at 3000 rpm for 6 minutes to separate the supernatant; add to each liter of supernatant 110ml of BaCl with a concentration of 1mol / l 2 Solution, centrifuge at 3800 rpm at 4°C for 5 minutes to collect the precipitate; add 0.2 mol / l EDTA solution with a pH value of 7.4 to the precipitate for desorption, and centrifuge at 3600 rpm at 4°C Centrifuge for 7 minutes to discard the precipitate and obtain the crude prothrombin solution; add CaCl to the crude prothrombin solution at room temperature 2 The solution makes the concentration of the crude prothrombin solution reach 0.1 mol / l, and then the pure thrombin product is obtained through DEAE-cellulose anion exchange colu...

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PUM

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Abstract

The thrombase separating and purifying process includes: adding sodium citrate solution as anticoagulant into animal blood as material; centrifugally separating to obtain plasma; freezing, defreezing and re-centrifuging to obtain supernatant; adding BaCl2 into supernatant, centrifuging and collecting precipitate; adding EDTA solution into precipitate to desorb, and centrifuging to eliminate precipitate and obtain coarse thrombin solution; adding CaCl2 solution to the coarse thrombin solution to make the coarse thrombin solution reach a concentration of 0.1 mol / L, chromatographic separation in DEAE-cellulose anionic exchange column to obtain pure thrombase product; and freeze drying to obtain the thrombase product. The present invention has high thrombase yield of 0.4 % and high thrombase purity, specific activity of 1500 U / mg protein.

Description

technical field [0001] The invention belongs to the method for separating and purifying thrombin in the technical field of bioengineering. Background technique [0002] The existing thrombin separation and purification process is relatively complicated: one is to add 32ml of BaCl with a concentration of 1mol / L dropwise to 400ml of fresh raw pig plasma 2 solution, stirred, and centrifuged to collect the precipitate. Precipitation with 0.02mol / L, pH7.4 Tris-HCl buffer, after washing twice, with 0.02mol / L, Tris-HCl buffer, pH7.4 (containing 0.2mol / l of EDTANa 2 , 0.1mol / LNaCl) to desorb, centrifuge to get the supernatant, and dialyze overnight. Solid (NH 4 ) 2 SO 4 To 35% saturation, to precipitate. Then the sample was added to the solid (NH 4 ) 2 SO 4 When a saturation of 65% to 70% is reached, the precipitate is collected by centrifugation. The precipitate was dissolved in 0.02mol / L Tris-HCl pH 8.0, and then dialyzed overnight. The dialyzed sample solution was adde...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74
Inventor 马永征李敏康宋宏新王旭
Owner SHAANXI UNIV OF SCI & TECH
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